Blood, 15 May 2001, Vol. 97, No. 10, pp. 3311-3312
BRIEF REPORT
Induction of tolerance to human factor VIII in mice
Hengjun Chao and
Christopher E. Walsh
From the University of North Carolina Gene Therapy
Center and the Department of Medicine, University of North Carolina at
Chapel Hill, Chapel Hill, NC.
 |
Abstract |
This paper reports loss of human factor VIII (hFVIII) inhibitory
antibody in immunocompetent C57BL/6 mice. High-titer anti-hFVIII antibody developed in the mice within 7 to 14 days of intraportal administration of adeno-associated virus (AAV) carrying FVIII that
coincided with a reduction in plasma hFVIII antigen. Bethesda titers
(> 100 units) persisted relatively unchanged for 9 to 10 months.
Unexpectedly, at 10 months after injection of the virus, hFVIII protein
(up to 59 ng/mL) was detected in 3 mice at the same time as
disappearance of hFVIII inhibitor. The level of hFVIII was similar to
that found in immunodeficient mice receiving the same dose of
recombinant AAV carrying hFVIII without hFVIII inhibitor. These results
suggest that tolerance to hFVIII can be induced by sustained expression
of hFVIII in a mouse model. Further elucidation of this observation may
affect use of FVIII gene transfer in the treatment of
inhibitor-positive patients with hemophilia A.
(Blood. 2001;97:3311-3312)
© 2001 by The American Society of Hematology.
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Introduction |
Gene therapy for hemophilias has become an
exciting prospect for long-term curative treatment.1
Advances in gene transfer for hemophilia A and B have shown the
feasibility of this curative procedure.2,3 However, the
immune response against human factor VIII (hFVIII) may be a difficult
barrier to successful gene therapy for hemophilia. Inhibitory
anti-hFVIII antibodies (inhibitors) develop in up to 30% of patients
with hemophilia A receiving infusions of factor VIII (FVIII)
concentrates.4-6 The mechanism responsible for development
of the anti-FVIII inhibitor remains unclear, although several factors
may affect its formation, including the type of FVIII mutation, the
FVIII product used, and patients' immune response.4-6
Currently, patients with the inhibitor are treated with induction of
immune tolerance7 or administration of porcine
FVIII,8 activated factor complexes,9 immunosuppressive agents,10 or activated factor
VIIa.11,12 Successful induction of immune
tolerance can be achieved by continuous infusion of a high-dose FVIII
concentrate to "desensitize" patients.7,11 Because of
the desirability of achieving long-term factor expression through gene
transfer, coupled with the potential of inhibitor development, we
performed experiments addressing inhibitor formation and tolerance in
gene transfer.
We previously reported sustained expression of hFVIII in mice with use
of recombinant adeno-associated virus (rAAV). When rAAV vector carrying
hFVIII complementary DNA was administered by the intraportal route into
immunocompetent C57BL/6 mice, high-titer anti-hFVIII inhibitor was
detected.3 These data are consistent with findings of
earlier studies in which inhibitor against hFVIII was detected in
immunocompetent mice receiving either repeated administration of
purified recombinant hFVIII protein or gene transfer of the hFVIII
gene.13,14 We here report that the antibody response to
hFVIII expressed from rAAV vector diminishes over time.
| |
Study design |
Vector constructs, cell cultures, rAAV production and
purification, the enzyme-linked immunosorbent assay (ELISA) for hFVIII antigen, and the essays for activated partial thromboplastin time, Coatest FVIII activity, and Bethesda inhibitor for hFVIII were as
described previously, as were the animal care and surgical procedures.3
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Results and discussion |
A total of 1010 to 1011 rAAV/DLZ6 virions
expressing functional B-domain-deleted (BDD) hFVIII were injected into
the portal vein of 4- to 6-week-old male C57BL/6 mice or nonobese
diabetic/severe combined immunodeficiency disease (NOD/SCID) mice. Up
to 27.5% (55 ng/mL) of the normal hFVIII antigen level was detected in the plasma of the NOD/SCID mice. However, maximum hFVIII levels in the
C57BL/6 mice were only 6 to 10 ng/mL. High-titer hFVIII inhibitor was
observed in plasma as early as 1 week after injection in all C57BL/6
mice given rAAV/DLZ6. Anti-hFVIII inhibitor titer increased to the
maximum level 9 to 12 weeks after injection.3
Six C57BL/6 mice were given an injection of rAAV/DLZ6. Three of the 6 mice were killed 9 months after administration of rAAV vectors for
performance of histologic and molecular analyses. The 3 surviving mice
were tested for hFVIII antigen at bimonthly intervals (it has now been
1.5 years since those mice received the rAAV/DLZ6 injection). At 10 months after injection, the hFVIII antigen level in 2 of the 3 mice
increased from a range of 3% to 5% to 29% (58 ng/mL) of the normal
level of hFVIII. Coincident with this rise in antigen levels was
disappearance of the anti-hFVIII inhibitor in plasma in the 3 mice
(Figure 1). These results are consistent
with previous observations of development of anti-hFVIII inhibitor in
either adult immunocompetent C57BL/6 mice or neonatal FVIII knockout
mice (C57BL/6 background) after repeated infusions of
hFVIII.13,14 The immature immune status of neonates or the relatively high FVIII levels may have accounted for the lower incidence
of inhibitor formation in those investigations.
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| Figure 1.
Level of hFVIII antigen and hFVIII inhibitor titer in
C57BL mice (n = 3) after administration of rAAV and BDD-hFVIII.
Solid line indicates hFVIII antigen level; and broken line,
anti-BDD-hFVIII inhibitor titer. The ELISA standard curve for hFVIII
was obtained by using pooled normal human plasma diluted in either
barbital-buffered saline or mouse plasma. Values (± SEM) represent the
averages of three animals.
|
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Our experimental data are consistent with observations of immune
tolerance in hemophilia patients with inhibitor. A wide range of doses
of FVIII are used clinically to induce tolerance; administration of
either high- or low-dose FVIII regimens is effective.15
Recombinant adenovirus, which is well-known to elicit cell-mediated
immune response, did not provoke an inhibitor response to hFVIII in
C57Bl/6 mice.16 Adenoviral production of supratherapeutic
amounts of hFVIII (5- to 10-fold greater than physiologic levels) may
facilitate immediate desensitization and immune tolerance that occurs
with repeated administration of high-dose FVIII to patients with
hemophilia A who have high-titer inhibitor.7,11
In another report,17 retroviral gene transfer and bone
marrow transplantation induced immunologic unresponsiveness in 50% of
FVIII knockout mice (C57BL/6 background). Although no hFVIII protein or
activity was detected, it was postulated that low-level exposure of
protein in antigen-presenting cells could induce tolerance. This
induction of central tolerance produced by means of hematopoietic chimerism allows tolerization of developing lymphocytes in the bone
marrow and thymus. In contrast, our results suggest that peripheral
tolerance of mature lymphocytes was induced by constant exposure of
hFVIII protein leading to B-cell and T-cell anergy.18 The
reemergence of FVIII also confirms that rAAV-mediated transduced hepatocytes are stably maintained and not eliminated by cytotoxic immune effector cells.19,20
Gene therapy holds the promise of radically changing the way in which
hemophilia is treated. Investigation of the mechanism responsible for
development of anti-hFVIII inhibitor in animal models is important not
only for preclinical evaluation of protocols for hemophilia A gene
therapy but also because it will contribute to the elucidation of
inhibitor formation and its treatment in patients with hemophilia.
Our results demonstrate that persistent expression of hFVIII in
immunocompetent mice mediates immune tolerance.
| |
Footnotes |
Submitted September 28, 2000; accepted January 19, 2001.
Funded by National Institutes of Health grant RO1 DK54419. C.E.W. is a
recipient of the Lucille Markey Trust.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Christopher E. Walsh, UNC Gene Therapy Center, Rm
7101, Thurston Bldg, CB 7352, University of North Carolina at Chapel
Hill, Chapel Hill, NC 27599; e-mail:cwalsh{at}med.unc.edu.
| |
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Abstract
Introduction
